PROJECT SUMMARY Hematopoietic malignancies result from dysregulated self-renewal pathways and an altered differentiation program. Traditional chemotherapy treatment is often used in the treatment of acute myeloid leukemia (AML) but approximately 30% of patients relapse within five years. Recent therapeutic approaches have focused on forcing leukemic cells to differentiate and in some cases have found great success. However, our understanding of the pathways that control differentiation is lacking. Despite the many studies focusing on genetic and epigenetic regulators in AML, there remains a gap in our understanding in how RNA transcripts are post-transcriptionally controlled. Recent discoveries from my sponsor's lab have found that mRNAs are subjected to the reversible base modification N6-methyladenosine (m6A) and this ?epitranscriptomic? regulation is a novel and potent regulator of many molecular processes including: transcript stability, mRNA splicing, and protein translation. Importantly, embryonic stem cells deficient in m6A remain pluripotent despite treatment with diverse stimuli that promote differentiation. On the other hand, more highly differentiated stem cells, such as primed stem cells, show enhanced and abnormal expression of differentiation markers upon depletion of m6A. These studies show that alterations in m6A levels can alter differentiation pathways. A major goal of this proposal is to test the innovative concept that m6A has critical roles in determining the differentiation of leukemia cells. My preliminary studies suggest that m6A ?writers? are highly expressed in myeloid leukemia cells and are required for maintaining leukemic cells in an undifferentiated state. Therefore, I hypothesize that aberrant m6A is a hallmark of leukemia and targeting the methylation machinery represents a new path forward for drug discovery. With the combined expertise of my sponsor and co-sponsor's laboratories I am ideally placed to address key questions to identify new differentiation-inducing strategies for AML: Is m6A necessary and sufficient for the development and progression of AML by controlling cellular differentiation? What mRNAs are aberrantly regulated by m6A in leukemia and how does m6A affect their function? Are there cellular pathways that are particularly susceptible to m6A levels? To address these questions I will (1) use established AML mouse models to define the functional requirement for m6A in leukemia formation and progression; and (2) uncover the m6A program that leads to leukemia by utilizing technological innovations developed in my sponsor's lab to generate nucleotide-level resolution of m6A in cells. I will create an m6A map in leukemia to identify leukemia-specific m6A sites and determine how m6A controls myeloid leukemia cell differentiation. Together, these experiments are poised to fundamentally alter how we view normal hematopoietic and leukemic differentiation and addresses whether m6A is a critical regulator of this process. These studies offer a new direction in the development of more effective differentiation-inducing therapies for AML. !